Electrostatic potential difference between tumor and paratumor regulates cancer stem cell behavior and prognose tumor spread

Abstract Tumor spread is responsible for most deaths related to cancer. Increasing the accuracy of cancer prognosis is critical to reducing the high mortality rates in cancer patients. Here, we report that the electrostatic potential difference (EPD) between tumor and its paratumor tissue is a prognostic marker for tumor spread. This finding is concluded from the patient‐specific EPD values and clinical observation. The electrostatic potential values were measured on tissue cryosections from 51 patients using Kelvin probe force microscopy (KPFM). A total of ~44% (15/34) patients of Vtumor–paratumor > 0 were featured with tumor spread, whereas only ~18% (2/11) patients of Vtumor–paratumor < 0 had tumor spread. Next, we found the increased enrichment of cancer stem cells in paratumors with lower electrostatic potentials using immunofluorescence imaging, which suggested the attribution of tumor spread to the galvanotaxis of cancer stem cells (CSCs) toward lower potential. The findings were finally validated in breast and lung spheroid models composed of differentiated cancer cells and cancer stem cells at the ratio of 1:1 and embedded in Matrigel dopped with negative‐, neutral‐ and positive‐charged polymers and CSCs prefer to spread out of spheroids to lower electrostatic potential sites. This work may inspire the development of diagnostic and prognostic strategies targeting at tissue EPDs and CSCs for tumor therapy.

Electric fields regulate cell proliferation, 4 differentiation, 5 migration, 6 and various other important biological processes, 7,8 though the underlying mechanisms remain unclear. 9,10 It has been suggested that exogenous electric fields can affect the cell's transmembrane potential, 11 by altering the activity of membrane ion channels. 12 Interestingly, highly proliferative cells have a significantly depolarized membrane potential compared to nonproliferating cells. 8 Additionally, membrane depolarization inhibits stem cell differentiation 4 and plays a crucial role in cytoskeletal rearrangement, 13 which is required for both mitosis and cell migration. Therefore, it is plausible that the inherently different bioelectric properties of the tumor microenvironment can promote the maintenance and spread of CSCs. For example, galvanotaxis of breast cancer cells (4T1) was demonstrated on the application of physiological levels of electric fields in vitro. 14 Electrical stimulation has been extensively investigated for its potential to modulate immune responses 15 and destroy cancer cells 16,17 (including CSCs 18 ). However, the use of electrostatic potentials for CSC identification, quantification, and migratory studies remains underexplored. Devising the bioelectric nature of CSCs and understanding their behavior under electrostatic gradients could provide physical markers [19][20][21][22][23] to enable a more robust distinction and preferential administration between CSCs and differentiated tumor cells. The integration of such physical markers into current practices for tumor evaluation may strengthen cancer prognosis and treatment.
Here, we investigated the correlation between tumor spread and tumor-to-paratumor EPD by using both patient tissues and in vitro models. We found that tumor cryosections with a positive potential difference correlated with higher rates of tumor spread in patients ( Figure 1) and higher expression of CD44-a known CSC marker. 24 We also demonstrated the directed migration of CSCs toward regions of low electrostatic potential in vitro. We, therefore, propose that the tumor-to-paratumor electrostatic potential difference (EPD), which can be measured on cryosections of tumor biopsy, is prognostic of tumor spread.

| Tumor-paratumor EPD shows a high correlation with chances of tumor spread
We hypothesized that tumor and paratumor tissues could have different electrostatic potentials in most types of tumors and that these potentials could be measured in cryosections and may correlate with tumor spread in patients. We, therefore, collected tumor and paratumor tissue samples from 51 patients, comprising 16 different types of cancers classified as Grades 1-3, some of which had and had not spread (Table S1). We first prepared tumor and paratumor tissue cryosections and measured the electrostatic potentials using a Kelvin probe force microscope (KPFM) 25 ( Figure S1). We then compared the electrostatic potentials between the tumor and paratumor regions and grouped the samples according to potential differences and F I G U R E 1 Schematic diagram depicting the finding that tumor spread is more likely to occur when the electrostatic potential between tumor and paratumor tissues (V Tumor-Paratumor ) is positive whether tumors had spread. These data suggest that tumors were more likely to spread when the electrostatic potential was higher in the  Figure 3). The C4.5 algorithm was used to perform fitting (parameter estimation). It is known from the previous step that the splitting threshold in a point inside the highlighted interval could reach optimal Gini impurity. The final splitting threshold would be the arithmetic average of boundary values. In this study, the splitting threshold became (2.5 + 3) Â 0.5 = 2.75. By using the same method, the splitting threshold for EPD was obtained at À11.8 and 50.65. We found that these values provided a high confidence correlation for tumor prognosis; for Group 1, all 8 (100%) tumors were reported nonspread, whereas, for Group 4, eight out of nine (89%) tumors had spread (Table S2). In Figure 3, the percentages of tumor spread in Groups 2 and 3 were 25% (6/24) and 40% (2/5), respectively. Groups 2 and 3 showed a weak correlation between tumor spread and V Tumor-Paratumor .
F I G U R E 2 Patient sample data and electrostatic potential differences. (a, b) Electrostatic potential differences between tumor and paratumor cryosections (V Tumor-Paratumor ) were measured using a Kelvin probe force microscope (KFPM). Samples with a positive, negative, or negligible potential difference are denoted by yellow, green, or gray dots, respectively. Data are presented as the mean value ± standard deviation of five measurements taken at different locations. (c) Samples are grouped according to the electrostatic potential difference.    I G U R E 3 Patient data grouped based on tumor grading and EPD (V tumor-paratumor ). By setting thresholds on grade 2.75 (a) and on potential differences À11.8 and 50.56 mV (b), the study cohorts were classified into four groups (1-4, C). Group (1, EPD < À11.8 mV) and Group (4, EPD > 50.65 mV) displayed high correlation between EPD and tumor spread respectively) from simulated spheroid models to the embedding charged Matrigel substrate.

| CSCs are enriched in paratumors of lower electrostatic potential
Our results show that tumor spread has a strong correlation with the EPD between tumor and paratumor, and patients with positive V Tumor-Paratumor have higher rates to develop tumor spread. This is consistent with a recent study that showed that high tumor potential value was correlated with the advanced stage of epithelial ovarian cancer. 28 Additionally, CD44+ CSCs showed directional migration toward the negatively charged substrate. We also observed that a higher abundance of CD44+ CSCs was observed in patient paratumor tissue when V Tumor-Paratumor was positive. CSCs present in metastatic tumors have a relatively depolarized (less negative) membrane potential as compared to differentiated cancer cells. 13   Samples were frozen and stored and shipped at À80 C to the laboratory for further processing.
A piece of gold was used as a reference. Each measurement was repeated five times at different locations.